Gene therapy is one of therapeutic methods that have been newly discovered to treat various diseases including cancers. The success of the gene therapy may be determined according to whether genes useful to treat target diseases are found and how efficiently the genes are delivered to target tissues. There are various gene delivery systems, and an adenoviral vector is currently being spotlighted as a carrier.
There are largely three reasons that adenoviruses are useful as a gene carrier: 1) the viral gene is not inserted into the host cell genome; 2) it can easily infect even non-dividing cells; and 3) it can efficiently deliver a target gene in vivo (Brody and Crystal, 1994). However, adenoviruses have one drawback regardless of the above advantages. The drawback is that replication competent adenovirus (RCA) may be produced upon production of recombinant adenoviruses.
Recombinant adenoviral vectors can be relatively easily mass-produced as compared with other virus vectors. The replication competence of the recombinant adenoviral vector can be simply lost by the deletion in early region (E1) of the viral genome. All adenoviral vectors may be this type of replication-incompetent vector. Conventionally, HEK293, a cell line having a part of the viral genome (nucleotides 1-4344), is used to construct the replication-incompetent recombinant adenoviral vector. Since E1 is crucial for the production of viruses, a cell line capable of temporarily supplying E1 is used. When the replication-incompetent adenoviruses are produced, only replication-incompetent viruses need to be produced. However, replication-competent adenoviruses may be produced due to the homologous recombination between the viral genome region except adenoviral E1 in HEK 293 cells and the viral genome region in the vector.
When the recombinant adenoviruses are produced, the production of RCA may cause the following problems: 1) the replication degree of viruses that are internally administered cannot be controlled; 2) replication-incompetent adenoviruses may be converted into replication-competent adenoviruses; and 3) the production of adenoviruses in an amount more than the amount required for gene therapy may cause tissue damage and severe toxicity. In order to solve these problems, many researchers who research recombinant adenoviral vectors have endeavored to develop adenovirus-producing cell lines capable of substituting for HEK293 cells and reducing the possibility of RCA production.
Since HEK293 cells contain considerably more regions of the adenoviral genome other than the adenovirus E1 gene, the possibility of the homologous recombination between HEK293 cells and the E1-deficient adenoviral vector may be high. Therefore, cells substituting for HEK293 cells have been developed to contain fewer regions of the adenoviral genome than HEK293 cells. During the 1990s, 911 cells were first developed as an adenovirus producing cell line. 911 cells are based on human derived retinoblasts. These cells have a part of the adenoviral genome (nucleotides 79-5789), and have excellent virus production capacity but similar RCA production capability as compared with HEK293 cells (Fallaux et al., 1996). Around the same time, an adenovirus producing cell lines using A549 cells, which are human bronchial cancer cells, were also developed. These cells were characterized by having fewer regions of the adenoviral genome (nucleotides 505-4034), and failed to be commercialized due to their poor adenoviral production capacity. PER.C6 cells are an adenovirus producing cell line that is the most widely known and commercially available, and developed based on human-derived retinoblasts like the above-described 911 cells. These cells are characterized by having fewer regions of the adenoviral genome (nucleotides 459-3510) as compared with the above-described cell lines. These cells exhibited a significant reduction in RCA production as compared with HEK293 cells. However, it has been reported that RCA was produced upon the mass production of this cell line (Murakami et al., 2004). Besides, adenovirus producing cells may be developed using human uterine cervical cancer cells, HeLa cells, but failed to be commercialized due to reasons such as, failing to significantly lower the possibility of RCA production, reducing the virus production capacity, and the like (Gao et al., 2000, Kim et al., 2001).
In order to solve the disadvantages of the above-described adenovirus producing cell lines, the present inventors have endeavored to develop adenovirus producing cell lines capable of: 1) remarkably lowering the possibility of RCA production by having fewer regions of adenoviral genome; and 2) having adenovirus production capacity superior to or similar to that of HEK293 cells.
Throughout this application, several patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications is incorporated into this application in order to more fully describe this invention and the state of the art to which this invention pertains.